(A) Intracellular H2O2 levels were assessed by flow cytometry after incubation with the redox-sensitive fluorescent dye DCF-DA. Shown is 1 representative patient sample and 1 representative normal control. (B) Mean intensity of DCF-DA fluorescence was compared between normal thymocytes (n = 7) and T-ALL (n = 5). Data are representative of most T-ALL samples (see Table 1) analyzed in 4 independent experiments. (C) TAIL7 cells previously treated with or without 1 mM H2O2 for 30 min were lysed in nonreducing conditions and analyzed for expression of PTEN oxidized and reduced bands. DTT was added where indicated to demonstrate the specificity of the lower, oxidized band. (D and E) PTEN-positive TAIL7, HPB-ALL, or primary T-ALL cells and PTEN-null Jurkat cells were treated for 2 h with 0.5 mM β-ME (D) or for 30 min with 1 mM H2O2 (E), and levels of Akt and GSK-3β phosphorylation were determined by immunoblot. (F) TAIL7 and HPB-ALL cells were pretreated for 1 h with 25 μm LY294002 (LY) or with DMSO vehicle control, stimulated with 1 mM H2O2 for 30 min, and analyzed for Akt phosphorylation by immunoblot. (G and H) Effects of β-ME (G) or H2O2 (H) on PTEN activity were measured by using the indirect PTEN redox assay, as described previously (19). More relative PTEN activity in the assay reflects less PTEN activity in the cell. Data from 2 independent experiments were normalized to the highest value in the control conditions. Values in B, G, and H are mean ± SEM.

Cooperative effects of combinatorial treatment of T-ALL cells with pharmacological antagonists of PI3K, CK2, and ROS.

(A and B) Inhibition of CK2 and ROS scavenging cooperate in inducing T-ALL cell death. TAIL7 cells were cultured with the indicated concentrations of TBB and β-ME (A), TBB and NAC (B), or their combination, and viability was assessed at 72 h. (C–F) Inhibition of PI3K signaling cooperates with inhibition of CK2 and ROS scavenging in inducing T-ALL cell death. TAIL7 cells were cultured with the indicated concentrations of LY294002 and TBB (C), LY294002 and DRB (D), LY294002 and β-ME (E), LY294002 and NAC (F), or their combination, and viability was assessed at 72 h. Percent viability relative to untreated control (vehicle alone) is indicated for each condition. Similar results were obtained using HPB-ALL and primary T-ALL cells (Supplemental Figures 17 and 18). Data are mean ± SEM.

Model for CK2- and ROS-mediated activation of the PI3K/Akt pathway in primary T-ALL.

By as-yet unknown mechanisms, CK2 is overexpressed and hyperactivated in T-ALL cells. Likewise, ROS intracellular levels are clearly elevated. These phenomena lead to posttranslational, nondeletional inactivation of PTEN, thereby contributing to constitutive hyperactivation of the PI3K/Akt pathway in leukemia cells. It is possible that CK2 and ROS may also mediate PTEN-independent activation of the PI3K/Akt pathway in some T-ALL cells.